A novel mass spectrometric epitope mapping approach without immobilization of the antibody
The development and application of a mass spectrometry-based epitope mapping procedure in solution and without immobilization of the antibody is described. Antigens were digested with proteases. Then size exclusion micro-column chromatography (SEC) was carried out prior to and upon exposure of the peptide mixtures to monoclonal antibodies. The epitope-containing peptide, affinity bound to the antibody and, thus, forming a stable complex eluted early as did all high-mass components, whereas all unbound low-mass peptides eluted late. Comparison of elution profiles in the presence and absence of the antibody showed a shift only for epitope-bearing peptides, enabling direct identification of the epitope. His-tag containing recombinant antigens Fibrillarin and RA33 were used in combination with an anti-His-tag monoclonal antibody in order to develop the method. Application of this method for the determination of an epitope on RA33 against which a monoclonal antibody was directed identified the epitope sequence (85IDGRVVEPKRA95) using MS/MS peptide sequencing. Advantages of this approach include low sample consumption, few handling steps, and short duration of analysis. With our method we are ultimately aiming at developing a screening procedure to identify major epitopes in patients that may be suitable in the future for stratification of patients, needed for personalized therapies. © 2011 El-Kased RF, et al.
El-Kased, Reham F.; Koy, C.; Lorenz, P.; Montgomery, H.; Tanaka, K.; Thiesen, H. J.; and Glocker, M. O., "A novel mass spectrometric epitope mapping approach without immobilization of the antibody" (2011). Pharmacy. 471.